Culture-free identification of fast-growing cyanobacteria cells by Raman-activated gravity-driven encapsulation and sequencing

By directly converting solar energy and carbon dioxide into biobased products, cyanobacteria are promising chassis for photosynthetic biosynthesis. To make cyanobacterial photosynthetic biosynthesis technology economically feasible on industrial scales, exploring and engineering cyanobacterial chassis and cell factories with fast growth rates and carbon fixation activities facing environmental stresses are of great significance. To simplify and accelerate the screening for fast-growing cyanobacteria strains, a method called Individual Cyanobacteria Vitality Tests and Screening (iCyanVS) was established. We show that the (13)C incorporation ratio of carotenoids can be used to measure differences in cell growth and carbon fixation rates in individual cyanobacterial cells of distinct genotypes that differ in growth rates in bulk cultivations, thus greatly accelerating the process screening for fastest-growing cells. The feasibility of this approach is further demonstrated by phenotypically and then genotypically identifying individual cyanobacterial cells with higher salt tolerance from an artificial mutant library via Raman-activated gravity-driven encapsulation and sequencing. Therefore, this method should find broad applications in growth rate or carbon intake rate based screening of cyanobacteria and other photosynthetic cell factories.